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Scaffolded Helical Bundles

Scaffolded helical bundles are what we typically think of when we say DNA origami. First created by Paul Rothemund in 20061. This class of structure is characterized by the folding of long DNA scaffolds into helical bundles packed side-by-side and most commonly arranged into either a honeycomb or square lattice. Each lattice arrangement has different pros and cons, and which to choose should depend on your intended application. Briefly:

1. Honeycomb Lattice

Each helix in a honeycomb has 3 possible neighbors arranged at 120 degree angles from each other. A DNA helix can crossover with one of its three neighbors at 7 bp intervals (corresponding to about 120 degrees of rotation) and with the same helix at 21 bp intervals (2 full turns of a DNA helix)23. The honeycomb lattice is the most popular for 3D origami and is thought to provide the most mechanically rigid arrangement. Compared to double stranded DNA, bundling of helices into a honeycomb pattern significantly increases the bending rigidity4. Per unit mass, honeycomb lattice creates origami with larger bending rigidity than square lattices due to a higher cross-section5. The honeycomb lattice cross over spacing complies with the natural B-form DNA twist density. Square lattice cross over spacing causes slight underwinding, and thus honeycomb lattices typically have less torsional strain than square lattices.

2. Square Lattice

Each DNA helix can have up to 4 neighbors which are arranged at 90 degree angles from each other. A DNA helix can crossover with one of its three neighbors at 8 bp intervals (corresponding to about 90 degrees of rotation) and with the same helix at 32 bp intervals (about 3 full turns of a DNA helix)6. This lattice arrangement is popular when flat surfaces are desired, like in 2D origami or origami being deposited on surfaces. The crossover spacing leads to a slight underwinding which can contribute to a right-handed global twist. Methods involving strategically placed insertions and deletions have been developed to compensate for and correct this twist7. Compared to the honeycomb lattice, the helical packing density is higher. Compared to honeycomb lattices, square lattices can achieve a greater density of addressable pixels8.

When designing your origami, other important considerations include scaffold choice, scaffold routing, staple routing, and crossover spacing.

Scaffold choice controls the size of your origami and also controls the sequence of your staples.

Scaffold routing can affect the yield, folding pathway, and structural stability of your origami9. As the scaffold is folded by staples there is an entropic penalty to confine the scaffold. Minimizing the entropic penalty of folding the scaffold and increasing cooperativity are two pathways thought to improve folding yield10. Generally this can be achieved by reducing the number of scaffold crossovers but careful consideration is required for each design11. The routing also affects structural stability as scaffold 'seams', regions of the origami where the scaffold crosses over, are mechanically weak points12.

Staple routing can also affect the yield, folding pathway, and structural stability of your origami. Similar to scaffold routing, consideration of your final intended function and origami shape are important when choosing staple routing. For improving yield of your origami, it is generally recommended to maximize the enthalpically favorable hybridization energy between staple and scaffold while minimizing the entropic penalty of scaffold loop closure. It is also important to ensure each domain of a staple is stable (>8 bp minimum). In addition, crossovers are considered mechanical weak points13, but crossover-rich regions often have less structural fluctuations12.

Crossover spacing can affect yield and structural properties. Increased crossover spacing can increase the structural rigidity of the origami, but it can also increase the internal strain. It can have functional consequences as well. Typically increased crossover densities require greater magnesium concentrations, but they are also less susceptible to nuclease degradation14.

References

  1. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).
  2. Reshetnikov, R. V. et al. A coarse-grained model for DNA origami. Nucleic Acids Res 46, 1102–1112 (2018).
  3. Castro, C. E. et al. A primer to scaffolded DNA origami. Nat Methods 8, 221–229 (2011).
  4. Kauert, D. J., Kurth, T., Liedl, T. & Seidel, R. Direct Mechanical Measurements Reveal the Material Properties of Three-Dimensional DNA Origami. Nano Lett. 11, 5558–5563 (2011).
  5. Shih, W. M. & Lin, C. Knitting complex weaves with DNA origami. Current Opinion in Structural Biology 20, 276–282 (2010).
  6. Ke, Y. et al. Multilayer DNA Origami Packed on a Square Lattice. J Am Chem Soc 131, 15903 (2009).
  7. Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into Twisted and Curved Nanoscale Shapes. Science 325, 725–730 (2009).
  8. Kilwing, L. et al. Multilayer DNA Origami with Terminal Interfaces That Are Flat and Wide-Area. ACS Nano 18, 885–893 (2024).
  9. DeLuca, M. et al. Mechanism of DNA origami folding elucidated by mesoscopic simulations. Nat Commun 15, 3015 (2024).
  10. Aksel, T., J. Navarro, E., Fong, N. & Douglas, S. M. Design principles for accurate folding of DNA origami. Proceedings of the National Academy of Sciences 121, e2406769121 (2024).
  11. Mohammadi-Zerankeshi, M., Houston, J., Elisha-Wigwe, O. K. U., Sachi, A. & Marras, A. E. Unraveling the Folding Dynamics of DNA Origami Structures. Small 21, e04855 (2025).
  12. Coarse-grained modelling of the structural properties of DNA origami. Nucleic Acids Research, https://academic.oup.com/nar/article/47/3/1585/5271501.
  13. Gerling, T., Kube, M., Kick, B. & Dietz, H. Sequence-programmable covalent bonding of designed DNA assemblies. Science Advances 4, eaau1157 (2018).
  14. Xin, Y. et al. Environment-Dependent Stability and Mechanical Properties of DNA Origami Six-Helix Bundles with Different Crossover Spacings. Small 18, 2107393 (2022).
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